CLK protein kinases and related products and methods

ABSTRACT

The present invention relates to nucleic acid molecules encoding mCLK2, mCLK3, and mCLK4 polypeptides, nucleic acid molecules-encoding portions of their amino acid sequences, nucleic acid vectors harboring such nucleic acid molecules, cells containing such nucleic acid vectors, purified polypeptides encoded by such nucleic acid molecules, and antibodies to such polypeptides. Also included are assays that contain at least one CLK protein kinase related molecule. Diagnosis and treatment of an abnormal condition related to RNA splicing or cell proliferation in an organism by using a CLK protein kinase related molecule or compound are disclosed. A method of using a CLK protein kinase related molecule or compound as a contraceptive to reproduction in male organisms is also disclosed.

RELATED APPLICATION

[0001] This application claims the benefit of PCT Application WO97/487.23 (PCT/IB97/00946), published on Dec. 24, 1997, filed on Jun.17, 1997, Axel Ullrich et al., entitled “Novel PTP20, PCP-2, BDP-1, CLK,and SIRP Proteins and Related Products and Methods,” and U.S.Provisional Patent Application Ser. No. 60/034,286, entitled “CLKProtein Kinases and Related Products and Methods,” Axel Ullrich andOliver Nayler, filed Dec. 19, 1996, both of which are incorporated byreference herein in their entirety including all figures, tables, anddrawings.

INTRODUCTION

[0002] The present invention relates to novel CDC2 like protein kinases(CLK protein kinases). These protein kinases phosphorylate proteins richin serine and arginine.

BACKGROUND OF THE INVENTION

[0003] The following description of the background of the invention isprovided to aid in understanding the invention, but is not admitted tobe or describe prior art to the invention.

[0004] Cellular signal transduction is a fundamental mechanism wherebyextracellular stimuli are relayed to the interior of cells andsubsequently regulate diverse cellular processes. One of the keybiochemical mechanisms of signal transduction involves the reversiblephosphorylation of proteins. Phosphorylation of polypeptides regulatesthe activity of mature proteins by altering their structure andfunction. Phosphate most often resides on the hydroxyl moiety (—OH) ofserine, threonine, or tyrosine amino acids in proteins. Enzymes thatmediate phosphorylation of cellular effectors fall into two classes.While protein phosphatases hydrolyze phosphate moieties from phosphorylprotein substrates, protein kinases transfer a phosphate moiety fromadenosine triphosphate to protein substrates. The converse functions ofprotein kinases and protein phosphatases balance and regulate the flowof signals in signal transduction processes.

[0005] Protein kinases and protein phosphatases are typically dividedinto two groups: receptor and non-receptor type proteins. Receptorprotein kinases are comprised of an extracellular domain, a membranespanning region, and a catalytic domain.

[0006] Protein kinases and protein phosphatases are divided further intothree classes based upon the amino acids they act upon. Some catalyzethe addition or hydrolysis of phosphate on serine or threonine only,some catalyze the addition or hydrolysis of phosphate on tyrosine only,and some catalyze the addition or hydrolysis of phosphate on serine,threonine, and tyrosine.

[0007] Membrane association is an important feature of signaltransduction. Protein kinases propagate extracellular signals to theinside of the cell by attracting other signaling molecules to themembrane. Schlessinger and Ullrich, 1992, Neuron 9:383-391. Forinstance, many receptor protein kinases bind an extracellular ligand,dimerize, and cross phosphorylate one another. These phosphate moietiessubsequently attract other proteins necessary for propagating the signalwithin the cell. The molecules that signal downstream of the receptorprotein kinases are often non-receptor protein kinases which propagateand amplify the extracellular signal.

[0008] A class of non-receptor protein kinases are implicated inregulating RNA splicing. Fu, 1995 RNA 1:663-680; Staknis and Reed, 1994,Mol. Cell. Biol. 14:7670-7682. These protein kinases phosphorylatepolypeptides rich in serine and arginine (SR proteins) SR proteins arecharacterized as containing at least one amino-terminal RNA recognitionmotif and a basic carboxy-terminal domain rich in serine and arginineresidues, often arranged in tandem repeats. Zahler et al., 1992, GenesDev 6:837-847. Experimental evidence supports the idea that the SRdomain is involved in protein-protein interactions (Kohtz et al., 1994,Nature 368:119-124) as well as protein-RNA interactions (Harada et al.,1996, Nature 380:175-179), and may contribute to a localization signaldirecting proteins to nuclear speckles. Hedley et al., 1995, Proc. Natl.Acad. Sci. USA 92:11524-11528.

[0009] A recent report demonstrated mCLK1, a CDC2 like kinase, interactswith ASF/SF2, SRp20 and hnRNP proteins in a yeast two hybrid system.Because hnRNP-K binds to the protooncogene p95^(vav), mCLK1 could beimplicated in transmitting signals that regulate the expression of theprotooncogenes myc and fos in hematopoietic cells. Furthermore, it wasdemonstrated, that mCLK1 could phosphorylate ASF/SF2 in vitro,suggesting, that SR containing proteins are the natural substrates ofmCLK1. Colwill et al., 1996, EMBO J. 15:265-275.

[0010] mCLK1 is a dual specificity protein kinase originally isolated inmouse expression libraries (Ben-David et al., 1991, EMBO J. 10:317-325;Howell et al., 1991, Mol. Cell. Biol. 11:568-572) and human (hCLK1,hCLK2, hCLK3), plant (AFC1, AFC2, AFC3) and fly (DOA) CLK proteinkinases have since been identified. Johnson and Smith, 1991, J. Biol.Chem. 266:3402-3407; Hanes et al., 1994, J. Mol. Biol. 244:665-672;Bender and Fink, 1994, Proc. Natl. Acad. Sci. USA 91:12105-12109; Yun etal., 1994, Genes. Dev. 8:1160-1173. The amino terminal domain of theseproteins is rich in serine and arginine, whereas the catalytic domaincan be most similar to CDC2, a serine/threonine protein kinase.Ben-David et al., 1991, EMBO J. 10:317-325.

[0011] Both mCLK1 and the Drosophila homologue, DOA, regulate RNAsplicing events. Each of these have two alternatively spliced productscoding for either the full-length catalytically active protein or atruncated protein lacking the catalytic domain. Yun et al., 1994, Genes.Dev. 8:1160-1173; Duncan et al., 1995, J. Biol. Chem. 270:21524-21531.Identical splice forms were also found in human CLK protein kinases.Hanes et al., 1994, J. Mol. Biol. 244:665-672. The ratio of these spliceproducts appears to be developmentally regulated in Drosophila (Yun etal., 1994, Genes. Dev. 8:1160-1173), and in a tissue and cell typespecific manner in mammals. Hanes et al., 1994, J. Mol. Biol.244:665-672; Duncan et al., 1995, J. Biol. Chem. 270:21524-21531. Inaddition, the expression of several other, larger transcripts, areobserved to be differentially regulated and are shown to representpartially spliced products. Duncan et al., 1995, J. Biol. Chem.270:21524-21531.

SUMMARY OF THE INVENTION

[0012] The present invention is based in part upon the isolation andcharacterization of nucleic acid molecules encoding CLK serine/threoninekinases designated mCLK2, mCLK3, and mCLK4. CLK serine/threonine kinasesregulate RNA splicing in cells and some are highly expressed in cancercells as well as testis. Various mCLK2, mCLK3, and mCLK4 relatedmolecules and compounds can now be designed as treatments of cancers oras contraceptives to reproduction in male organisms.

[0013] The present invention is based in part upon rucleic acidmolecules encoding novel mCLK2, mCLK3, and mCLK4 polypeptides, nucleicacid molecules encoding portions of their amino acid sequences, nucleicacid vectors harboring such nucleic acid molecules, cells containingsuch nucleic acid vectors, purified polypeptides encoded by such nucleicacid molecules, and antibodies to such polypeptides, and methods ofidentifying compounds that bind mCLK2, mCLK3, and mCLK4 or abrogatetheir interactions with natural binding partners. Also disclosed aremethods for diagnosing and treating specific abnormal conditions in anorganism with mCLK2, mCLK3, and mCLK4 related molecules or compounds.The nucleic acid molecules, nucleic acid vectors, recombinant cells,polypeptides, and antibodies may be produced using well known andstandard techniques used currently in the art.

[0014] Thus in a first aspect, the invention features isolated,enriched, or purified nucleic acid molecules encoding a novel mCLK2,mCLK3, or mCLK4 polypeptide.

[0015] The term “isolated”, in reference to nucleic acid molecules,indicates that a naturally occurring sequence has been removed from itsnormal cellular environment. The isolated nucleic acid of the presentinvention is unique in the sense that it is not found in a pure orseparated state in nature. Use of the term “isolated” indicates that anaturally occurring sequence has been removed from its normal cellular(i.e., chromosomal) environment. Thus, the sequence may be in acell-free solution or placed in a different cellular environment. Theterm does not imply that the sequence is the only nucleotide chainpresent, but that it is essentially free (about 90-95% pure at least) ofnon-nucleotide material naturally associated with it, and thus isdistinguished from isolated chromosomes.

[0016] The term “enriched”, in reference to nucleic acid molecules,means that the specific DNA or RNA sequence constitutes a significantlyhigher fraction (2-5 fold) of the total DNA or RNA present in the cellsor solution of interest than in normal or diseased cells or in the cellsfrom which the sequence was taken. A person skilled in the art couldenrich a nucleic acid mixture by preferentially reducing the amount ofother DNA or RNA present, or preferentially increasing the amount of thespecific DNA or RNA, or both. However, nucleic acid molecule enrichmentdoes not imply that there is no other DNA or RNA present, the term onlyindicates that the relative amount of the sequence of interest has beensignificantly increased. The term “significantly” qualifies “increased”to indicate that the level of increase is useful to the personperforming the recombinant DNA technique, and generally means anincrease relative to other nucleic acids of at least 2 fold, or morepreferably at least 5 to 10 fold or more. The term also does not implythat there is no DNA or RNA from other sources. Other DNA may, forexample, comprise DNA from a yeast or bacterial genome, or a cloningvector. In addition, levels of mRNA may be naturally increased relativeto other species of mRNA when working with viral infection or tumorgrowth techniques. This term distinguishes from naturally occurringevents, such as viral infection, or tumor type growths, in which thelevel of one mRNA may be naturally increased relative to other speciesof MRNA. That is, the term is meant to cover only those situations inwhich a person has intervened to elevate the proportion of the desirednucleic acid.

[0017] It is also advantageous for some purposes that a nucleotidesequence be in purified form. The term “purified” in reference tonucleic acid does not require absolute purity (such as a homogeneouspreparation). Instead, it represents an indication that the sequence isrelatively more pure than in the natural environment (compared to thenatural level this level should be at least 2-5 fold greater, e.g., interms of mg/mL). Individual clones isolated from a cDNA library may bepurified to electrophoretic homogeneity. The claimed DNA moleculesobtained from these clones could be obtained directly from total DNA orfrom total RNA. The cDNA clones are not naturally occurring, but ratherare preferably obtained via manipulation of a partially purifiednaturally occurring substance (messenger RNA). The construction of acDNA library from mRNA involves the creation of a synthetic substance(cDNA) and pure individual CDNA clones can be isolated from thesynthetic library by clonal selection of the cells carrying the cDNAlibrary. Thus, the process, which includes the construction of a cDNAlibrary from mRNA and isolation of distinct cDNA clones, yields anapproximately 10⁶-fold purification of the native message. Thus,purification of at least one order of magnitude, preferably two or threeorders, and more preferably four or five orders of magnitude isexpressly contemplated.

[0018] The term “nucleic acid molecule” describes a polymer ofdeoxyribonucleotides (DNA) or ribonucleotides (RNA). The nucleic acidmolecule may be isolated from a natural source by cDNA cloning orsubtractive hybridization or synthesized manually. The nucleic acidmolecule may be synthesized manually by the triester synthetic method orby using an automated DNA synthesizer. “cDNA cloning” techniques referto hybridizing a small nucleic acid molecule, a probe, to genomic cDNAthat is bound to a membrane. The probe hybridizes (binds) tocomplementary sequences of cDNA. The term “complementary” describes twonucleotides that can form multiple favorable interactions with oneanother. For example, adenine is complementary to thymidine as they canform two hydrogen bonds. Similarly, guanine and cytosine arecomplementary since they can form three hydrogen bonds. cDNAs aremolecules that are reverse transcribed from fragments of message RNAfrom a genomic source. These fragments form a CDNA library of nucleicacid molecules. cDNA libraries are constructed from natural sources suchas mammalian blood, semen, or tissue.

[0019] The term “subtractive hybridization” refers to a method similarto cDNA-cloning except that cDNA prepared from mRNA in unstimulatedcells is added to mRNA in stimulated or different types of cells.cDNA/mRNA can then be precipitated to enrich the mRNA specific to thestimulation signal or different cell.

[0020] The term “hybridize” refers to a method of interacting a nucleicacid probe with a DNA or RNA molecule in solution or on a solid support,such as cellulose or nitrocellulose. If a nucleic acid probe binds tothe DNA or RNA molecule with high affinity, it is said to “hybridize” tothe DNA or RNA molecule. As mentioned above, the strength of theinteraction between the probe and its target can be assessed by varyingthe stringency of the hybridization conditions. Various low or highstringency hybridization conditions may be used depending upon thespecificity and selectivity desired. Stringency is controlled by varyingsalt or denaturant concentrations. Under stringent hybridizationconditions only highly complementary nucleic acid sequences hybridize.Preferably, such conditions prevent hybridization of nucleic acidshaving one or two mismatches out of 20 contiguous nucleotides.

[0021] By “novel” is meant new and in the particular context of thepresent invention refers to CLK sequences that have not been previouslydescribed. In preferred embodiments the novel sequence may be thefull-length serine CLK2 or CLK3 sequence, the full-length mammalian CLK4sequence, or shorter fragments (preferably functional fragments) if anyof the above as long as they were not already previously described.

[0022] The terms “mCLK2”, “mCLK3”, and “mCLK4”refer to polypeptides thathave amino acid sequences substantially similar to those set forth inFIG. 1, FIG. 2, FIG. 4, or FIG. 6. A sequence that is substantiallysimilar will preferably have at least 95% identity, more preferably atleast 96-97% identity, and most preferably 98-100% identity to thesequence set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6. CLK proteinkinase polypeptides preferably have protein kinase activity andfragments of the full length CLK protein kinase sequences having suchactivity may be identified using techniques well known in the art, suchas sequence comparisons and assays such as those described in theexamples herein. Other aspects of mCLK2, mCLK3, and mCLK4 nucleic acidsequences, amino acid sequences, functions and properties are furtherdepicted in Nayler et al., 1997, Biochem J. 326: 693-700, herebyincorporated by reference herein in its entirety including all figures,tables, and drawings.

[0023] By “identity” is meant a property of sequences that measurestheir similarity or relationship. Generally speaking, identity ismeasured by dividing the number of identical residues by the totalnumber of residues and gaps and multiplying the product by 100. “Gaps”are spaces in an alignment that are the result of additions or deletionsof amino acids. Thus, two copies of exactly the same sequence have 100%identity, but sequences that are less highly conserved, and havedeletions, additions, or replacements, may have a lower degree ofidentity. Those skilled in the art will recognize that several computerprograms are available for determining sequence identity.

[0024] A preferred embodiment of the invention concerns nucleic acidmolecules relating to mCLK2, mCLK3, and mCLK4 that are enriched,isolated, or purified from a mammalian source. These nucleic acidmolecules can be isolated from, among others, blood, semen, or tissue.Although mCLK2, mCLK3, and mCLK4 nucleic acid molecules are isolatedfrom mouse cells, current recombinant DNA techniques can readilyelucidate related nucleic acid molecules in other mammalian tissue.Mammals include, but are not limited to, mice, rats, rabbits, cows,horses, monkeys, apes, and preferably humans.

[0025] Another preferred embodiment of the invention concerns isolatednucleic acid molecules that encode at least seventeen amino acids of amCLK2, mCLK3, or mCLK4 polypeptide. Preferably, at least 17, 20, 25, 30,35, 40, 50, 100, 200, 300, 400, 450, 475, or 485 contiguous amino acidsare encoded. This preferred embodiment of the invention is achieved byapplying routine recombinant DNA techniques known to those skilled inthe art.

[0026] Another aspect of the invention features a nucleic acid probethat can detect nucleic acid molecules encoding a mCLK2, mCLK3, or mCLK4polypeptide in a sample.

[0027] The term “nucleic-acid probe” refers to a nucleic acid moleculethat is complementary to and can bind a nucleic acid sequence encodingthe amino acid sequence substantially similar to that set forth in FIG.1, FIG. 2, FIG. 4, or FIG. 6.

[0028] The nucleic acid probe or its complement encodes any one of theamino acid molecules set forth in the invention. Thus the nucleic acidprobe can encode at least 17, 20, 25, 30, 35, 40, 50, 100, 200, 300,400, 450, 475, or 485 contiguous amino acids of the full-length sequenceset forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6.

[0029] The nucleic acid probe can be labeled with a reporter molecule ormolecules. The term “reporter molecule” refers to a molecule that isconjugated to the nucleic acid probe or is contained within the nucleicacid probe. The reporter molecule allows the detection of the probe bymethods used in the art. Reporter molecules are chosen from, but notlimited to, the group consisting of an enzyme, such as a peroxidase, aradioactive element, or an avidin or biotin molecule.

[0030] A nucleic acid probe, whether labeled or unlabeled, shouldhybridize to a complement in a sample. Various low or high stringencyhybridization conditions may be used depending upon the specificity andselectivity desired. Stringency is controlled by varying salt ordenaturant concentrations. Under stringent hybridization conditions onlyhighly complementary nucleic acid sequences hybridize. Preferably, suchconditions prevent hybridization of nucleic acid molecules having one ortwo mismatches out of 20 contiguous nucleotides, more preferably preventhybridization of nucleic acid molecules having one mismatch out of 35contiguous nucleotides, and most preferably prevent hybridization ofnucleic acid molecules having one mismatch out of 50 contiguousnucleotides.

[0031] The nucleic acid probe or complement can also refer to a nucleicacid molecule encoding a conserved or unique region of amino acids.These nucleic acid molecules are useful as hybridization probes toidentify and clone additional polypeptides relating to CLKserine/threonine kinases. The term “conserved nucleic acid regions”refers to regions present in two or more nucleic acid molecules encodinga CLK protein kinase polypeptide, to which a particular nucleic acidsequence can hybridize under lower stringency conditions. Examples oflower stringency conditions suitable for screening nucleic acidmolecules are provided in Abe, et al. J. Biol. Chem., 19:13361 (1992)(hereby incorporated by reference herein in its entirety, including anydrawings). Preferably, conserved regions differ by no more than 5 out of20 nucleotides, more preferably conserved regions differ by no more than10 out of 20 nucleotides, and most preferably conserved regions differby no more than 15 out of 20 nucleotides. Protein kinases shareconserved regions in the catalytic domain.

[0032] The term “unique nucleic acid region” concerns a sequence presentin a full length nucleic acid molecule encoding a CLK protein kinasepolypeptide that is not present in a sequence encoding any othernaturally occurring polypeptide. Such regions preferably comprise 30 or45 contiguous nucleotides, more preferably 100 contiguous nucleotides,and most preferably 200 contiguous nucleotides present in the fulllength nucleic acid sequence encoding a CLK protein kinase polypeptide.In particular, a unique nucleic acid region is preferably of mammalianorigin.

[0033] Methods for using the probes include detecting the presence oramount of CLK protein kinase RNA in a sample by contacting the samplewith a nucleic acid probe under conditions such that hybridizationoccurs and detecting the presence or amount of the probe bound to CLKRNA. The nucleic acid duplex formed between the probe and a nucleic acidsequence encoding a CLK protein kinase polypeptide may be used in theidentification of the sequence of the nucleic acid detected (for examplesee, Nelson et al., in Nonisotopic DNA Probe Techniques, p. 275 AcademicPress, San Diego (Kricka, ed., 1992) hereby incorporated by referenceherein in its entirety, including any drawings). Kits for performingsuch methods may be constructed to include a container holding a nucleicacid probe.

[0034] In yet another aspect, the invention relates to a nucleic acidvector comprising a promoter element and a nucleic acid moleculedescribed in the first aspect of the invention.

[0035] The term “nucleic acid vector” relates to a single or doublestranded circular nucleic acid molecule that can be transfected ortransformed into cells and replicate independently or within a cellgenome. A vector can be cut and thereby linearized upon treatment withrestriction enzymes. An assortment of vectors, restriction enzymes, andthe knowledge of the nucleotide sequences that the restriction enzymesoperate upon are readily available to those skilled in the art. Anucleic acid molecule encoding a CLK protein kinase can be inserted intoa vector by cutting the vector with restriction enzymes and ligating thetwo pieces together.

[0036] The term “promoter element” describes a nucleotide sequence thatis incorporated into a vector that, once inside an appropriate cell, mayfacilitate transcription factor and/or polymerase binding and subsequenttranscription of portions of the vector DNA into mRNA. The promoterelement precedes the SI end of the nucleic acid molecule of the firstaspect of the invention such that the latter is transcribed into mRNA.Recombinant cell machinery then translates mRNA into a polypeptide.

[0037] Many techniques are available to those skilled in the art tofacilitate transformation or transfection of the nucleic acid vectorinto a prokaryotic or eukaryotic organism. The terms “transformation”and “transfection” refer to methods of inserting a nucleic acid vectorinto a cellular organism. These methods involve a variety of techniques,such as treating the cells with high concentrations of salt, an electricfield, or detergent, to render the cell outer membrane or wall permeableto nucleic acid molecules of interest.

[0038] A nucleic acid vector can be useful for identifying naturalbinding partners of CLK serine/threonine kinases.

[0039] The term “natural binding partners” refers to polypeptides thatbind to CLK serine/threonine kinases and play a role in propagating asignal in a signal transduction process. The term “natural bindingpartner” also refers to a polypeptide that binds to CLK serine/threoninekinases within a cellular environment with high affinity. High affinityrepresents an equilibrium binding constant on the order of 10-1 M.However, a natural binding partner can also transiently interact with aCLK protein kinase and chemically modify it. CLK protein kinase naturalbinding partners are chosen from a group consisting of, but not limitedto, src homology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosinebinding domains, and receptor and non-receptor protein kinases orprotein, phosphatases.

[0040] Methods are readily available in the art for identifying bindingpartners of polypeptides of interest. These methods include screeningcDNA libraries included in one nucleic acid vector with a nucleic acidmolecule encoding the desired polypeptide in another nucleic acidvector. Vojtek et al., 1993, Cell 74:205214. These techniques oftenutilize yeast recombinant cells. These techniques also utilize twohalves of a transcription factor, one half that is fused to apolypeptide encoded by the cDNA library and the other that is fused tothe polypeptide of interest. Interactions between a polypeptide encodedby the cDNA library and the polypeptide of interest are detected whentheir interaction concomitantly brings together the two halves into anactive transcription factor which in turn activates a gene that reportsthe interaction. Any of the nucleic molecules encoding mCLK2, mCLK3, ormCLK4 can be readily incorporated into an nucleic acid vector used insuch a screening procedure by utilizing standard recombinant DNAtechniques in the art.

[0041] Another aspect of the invention relates to a recombinant cell ortissue comprising a nucleic acid molecule encoding a mCLK2, mCLK3, ormCLK4 polypeptide.

[0042] The term “recombinant” refers to an organism that has a newcombination of genes or nucleic acid molecules. A new combination ofgenes or nucleic acid molecules can be introduced to an organism using awide array of nucleic acid manipulation techniques available to thoseskilled in the art.

[0043] The recombinant cell can be a eukaryotic or prokaryotic organism.The term “eukaryote” refers to an organism comprised of cells containinga nucleus. Eukaryotes are differentiated from “prokaryotes” which do nothouse their genomic DNA inside a nucleus. Prokaryotes includeunicellular organisms such as bacteria while eukaryotes are representedby yeast, invertebrates, and vertebrates.

[0044] The recombinant cell can also harbor a nucleic acid vector thatis extragenomic. The term “extragenomic” refers to a nucleic acid vectorwhich does not integrate into a cell genome. Many nucleic acid vectorsare designed with their own origins of replication which allow them toutilize the recombinant cell replication machinery to copy and propagatethe nucleic acid vector nucleic acid sequence. These nucleic acidvectors are small enough that they are not likely to harbor nucleic acidsequences homologous to genomic sequences of the recombinant cell. Thusthese nucleic acid vectors replicate independently of the genome and donot recombine with or integrate into the genome.

[0045] A recombinant cell can also harbor a portion of a nucleic acidvector in an intragenomic fashion. The term “intragenomic” defines anucleic acid vector that integrates within a cell genome. Multiplenucleic acid vectors available to those skilled in the art containnucleic acid sequences that are homologous to nucleic acid sequences ina particular organism's genomic DNA. These homologous sequences willresult in recombination events that incorporate portions of the nucleicacid vector into the genomic DNA. Those skilled in the art can controlwhich nucleic acid sequences of the nucleic acid vector integrate intothe cell genome by flanking the portion to be integrated into the genomewith homologous sequences in the nucleic acid vector.

[0046] In yet another aspect, the invention features an isolated,enriched, or purified polypeptide encoded by a mCLK2, mCLK3, or mCLK4nucleic acid molecule of the invention.

[0047] The term “isolated”, in reference to a polypeptide, describes apolymer of amino acids conjugated to each other that are separated froma natural source. The polypeptide can also be synthesized manually.Isolated peptides can be at least 17, 20, 25, 30, 35, 40, 50, 100, 200,or 300 contiguous amino acids of one of the full-length sequences setforth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6. In certain aspects longerpolypeptides are preferred, such as those with 400, 450, 475, or 485 ofthe contiguous amino acids of mCLK2, mCLK3, or mCLK4 set forth in FIG.1, FIG. 2, FIG. 4, or FIG. 6.

[0048] The isolated polypeptides of the present invention are unique inthe sense that they are not found in a pure or separated state innature. Use of the term “isolated” indicates that a naturally occurringsequence has been removed from its normal cellular environment. Thus,the sequence may be in a cell-free solution or placed in a differentcellular environment. The term does not imply that the sequence is theonly amino acid chain present, but that it is essentially free (about90-95% pure at least) of non-amino acid material naturally associatedwith it.

[0049] The term “enriched”, in reference to a polypeptide, defines aspecific amino acid sequence constituting a significantly higherfraction (2-5 fold) of the total of amino acids present in the cells orsolution of interest than in normal or diseased cells or in the cellsfrom which the sequence was separated. A person skilled in the art canpreferentially reduce the amount of other amino acid sequences present,or preferentially increase the amount of specific amino acid sequencesof interest, or both. However, the term “enriched” does not imply thatthere are no other amino acid sequences present. Enriched simply meansthe relative amount of the sequence of interest has been significantlyincreased. The term “significant” indicates that the level of increaseis useful to the person making such an increase. The term also means anincrease relative to other amino acids of at least 2 fold, or morepreferably at least 5 to 10 fold, or even more. The term also does notimply that there are no amino acid sequences from other sources. Othersource amino acid sequences may, for example, comprise amino acidsequences from a recombinant organism. “Enriched” is meant to cover onlythose situations in which a person has intervened to elevate theproportion of the desired amino acid sequence.

[0050] The term “purified”, in reference to a polypeptide, does notrequire absolute purity (such as a homogeneous preparation); instead, itrepresents an indication that the amino acid sequence is relatively morepure than in a cellular environment. The concentration of the preferredamino acid sequence should be at least 2-5 fold greater (in terms ofmg/ml) than its concentration in a cellular environment. Purification ofat least one order of magnitude, preferably two or three orders, andmore preferably four or five orders of magnitude is preferred. Thesubstance is preferably free of contamination, as indicated by puritylevels of 90%, 95%, or 99%.

[0051] A preferred embodiment of the invention relates to a mCLK2,mCLK3, or mCLK4 polypeptide that is a unique fragment. This uniquefragment can contain at least 17, 20, 25, 30, 35, 40, 50, 100, 200, 300,400, 450, 475, or 485 contiguous amino acids of one of the full-lengthsequences. In addition, preferred lengths and portions of mCLK2, mCLK3,or mCLK4 amino acid sequences are encoded by the nucleic acid moleculesdefined in the first aspect of the invention.

[0052] The term “unique fragment” refers to the minimum stretch of aminoacids in one mCLK molecule that is different in sequence than any otherportion of another protein kinase. Since the largest identical stretchof amino acids found in FIG. 1, FIG. 2, FIG. 4, or FIG. 6 is seventeenamino acids, the minimum unique fragment for a mCLK protein kinase isseventeen amino acids.

[0053] The polypeptide can be isolated, enriched, or purified from aprokaryotic or eukaryotic recombinant cell. A eukaryotic cell includesmammals and preferably humans. Multiple standard techniques areavailable to those skilled in the art to facilitate isolation,enrichment, or purification of a polypeptide from recombinant cells.These methods typically include lysing the recombinant cells andseparating the polypeptide of interest from the rest of the cellpolypeptides, nucleic acids, and fatty acid-based material usingstandard chromatography techniques known in the art.

[0054] Another aspect of the invention features an antibody, that ismonoclonal or polyclonal, or an antibody fragment having specificbinding affinity to a mCLK2, mCLK3, or mCLK4 polypeptide.

[0055] Antibodies or antibody fragments are polypeptides with regionsthat can bind to other polypeptides with high affinity. The term“specific binding affinity” describes an antibody that binds to a mCLK2,mCLK3, or mCLK4 polypeptide with greater affinity than it binds to otherpolypeptides under specified conditions.

[0056] The term “polyclonal” refers to a mixture of antibodies withspecific binding affinity to a mCLK2, mCLK3, or mCLK4 polypeptide, whilethe term “monoclonal” refers to one antibody with specific bindingaffinity to a mCLK2, mCLK3, or mCLK4 polypeptide. A monoclonal antibodybinds to one specific region on a mCLK2, mCLK3, or mCLK4 polypeptide anda polyclonal mixture of antibodies can bind multiple regions of a mCLK2,mCLK3, or mCLK4 polypeptide. One skilled in the art would note that amonoclonal and especially a polyclonal antibody that has specificbinding affinity to a mCLK2, mCLK3, or mCLK4 polypeptide will mostlikely also have specific binding affinity to another CLK protein kinasepolypeptide of mammalian origin.

[0057] The term “antibody fragment” refers to a portion of an antibody,often the hypervariable region and portions of the surrounding heavy andlight chains, that displays specific binding affinity for a particularmolecule. A hypervariable region is a portion of an antibody thatphysically binds to the ligand to which it binds specifically.

[0058] Antibodies or antibody fragments having specific binding affinityto a mCLK2, mCLK3, or mCLK4 polypeptide may be used in methods fordetecting the presence and/or amount of a CLK protein kinase polypeptidein a sample by probing the sample with the antibody under conditionssuitable for CLK protein kinase-antibody immunocomplex formation anddetecting the presence and/or amount of the antibody conjugated to a CLKprotein kinase polypeptide. Diagnostic kits for performing such methodsmay be constructed to include antibodies or antibody fragments specificfor a CLK protein kinase as well as a conjugate of a binding partner ofthe antibodies or the antibodies themselves.

[0059] Another aspect of the invention features a hybridoma whichproduces an antibody having specific binding affinity to a mCLK2, mCLK3,or mCLK4 polypeptide. A “hybridoma” is an immortalized cell line whichis capable of secreting an antibody, for example an antibody withspecific binding affinity to a mCLK2, mCLK3, or mCLK4 polypeptide.

[0060] Another aspect of the invention relates to an isolated, enriched,or purified nucleic acid molecule comprising a nucleotide sequence that:(a) encodes a full length mCLK2, mCLK3, or mCLK4 amino acid sequence asset forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6; (b) encodes thecomplement of the nucleotide sequence encoding the amino acid sequencesof FIG. 1, FIG. 2, FIG. 4, or FIG. 6; (c) hybridizes under highlystringent conditions to the nucleic acid molecule of (a) and encodes anaturally occurring mCLK2, mCLK3, or mCLK4 protein; (d) a mCLK2, mCLK3,or mCLK4 protein having the full length amino acid sequence as set forthin FIG. 1, FIG. 2, FIG. 4, or FIG. 6 except that it lacks one or more ofthe following segments of amino acid residues 1-182, 183-470, or 471499of mCLK2, 1-176, 177-473, or 474-496 of mCLK3, or 1183, 184-486, or486-489 of mCLK4; (e) the complement of the nucleotide sequence of (d);(f) a polypeptide having the amino acid sequence set forth in FIG. 1,FIG. 2, FIG. 4, or FIG. 6 from amino acid residues 1-182, 183-470, or471-499 of mCLK2, 1176, 177-473, or 474-496 of mCLK3, or 1-183, 184-486,or 486-489 of mCLK4;(g) the complement of the nucleotide sequence of(f); (h) encodes a polypeptide having the full length amino acidsequence set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6 except that itlacks one or more of the domains selected from the group consisting of aN-terminal domain, a catalytic domain, and a C-terminal-region; or (i)the complement of the nucleotide sequence of (h).

[0061] The term “N-terminal domain” refers to a portion of the fulllength mCLK2, mCLK3, or mCLK4 amino acid sequences spanning from theamino terminus to the start of the catalytic domain.

[0062] The term “catalytic domain” refers to a portion of the fulllength mCLK2, mCLK3, or mCLK4 amino acid molecules that does not containthe N-terminal domain or C-terminal region and has catalytic activity.

[0063] The term “C-terminal region” refers to a portion of the fulllength mCLK2, mCLK3, or mCLK4 amino acid molecules that begins at theend of the catalytic domain and ends at the carboxy terminal amino acid,which is the last amino acid encoded before the stop codon in thenucleic acid sequence.

[0064] Domains are regions of polypeptides which contain particularfunctions. For instance, N-terminal or C-terminal domains of signaltransduction proteins can serve functions including, but not limited to,binding molecules that localize the signal transduction molecule todifferent regions of the cell or binding other signaling moleculesdirectly responsible for propagating a particular cellular signal. Somedomains can be expressed separately from the rest of the protein andfunction by themselves, while others must remain part of the intactprotein to retain function. The latter are termed functional regions ofproteins and also relate to domains.

[0065] Functional regions of mCLK2, mCLK3, or mCLK4 may be identified byaligning their amino acid sequences with amino acid sequences of otherpolypeptides with known functional regions. If regions of mCLK2, mCLK3,or mCLK4 share high amino acid identity with the amino acid sequences ofknown functional regions, then mCLK2, mCLK3, or mCLK4 can be determinedto contain these functional regions by those skilled in the art. Thefunctional regions can be determined, for example, by using computerprograms and sequence information available to those skilled in the art.

[0066] Other functional regions of signal transduction molecules thatmay exist within mCLK2, mCLK3, or mCLK4 include, but are not limited to,proline-rich regions or phosphoryl tyrosine regions. These regions caninteract with natural binding partners such as SH2 or SH3 domains ofother signal transduction molecules.

[0067] Another aspect of the invention relates to nucleic acid vectorscomprising any of the nucleic acid molecules described herein.

[0068] In another aspect, the invention includes recombinant cells ortissues comprising any of the nucleic acid molecules described herein.

[0069] In yet another aspect, the invention relates to a method ofidentifying compounds capable of inhibiting or activating CLK proteinkinase phosphorylation activity. This method comprises the followingsteps: (a) adding a compound to a mixture comprising a CLK proteinkinase polypeptide and a substrate for a CLK protein kinase; and (b)detecting a change in phosphorylation of said substrate.

[0070] The term “compound” includes small organic molecules including,but not limited to, oxindolinones, quinazolines, tyrphostins,quinoxalines, and extracts from natural sources.

[0071] The term “CLK protein kinase polypeptide” refers to any CLKprotein kinase isolated from an insect or a mammal. The polypeptide canbe the full length amino acid sequence (the contiguous amino acidsencoded by the nucleic acids spanning from the start codon to the stopcodon of a naturally occurring CLK protein kinase nucleic acid molecule)or portions of a naturally occurring full length CLK protein kinase.Preferably, at least 17, 20, 25, 30, 35, 40, 50, 100, 200, 300, 400,450, 475, or 485 contiguous amino acids are encoded for the CLK proteinkinase polypeptide.

[0072] The term “a change in phosphorylation”, in the context of theinvention, defines a method of observing a change in phosphorylation ofthe substrate in response to adding a compound to cells. Thephosphorylation can be detected, for example, by measuring the amount ofa substrate that is converted to a product with respect to time.Addition of a compound to cells expressing a CLK protein kinasepolypeptide may either enhance (activate) or lower (inhibit) thephosphorylation. If a compound lowers phosphorylation, the compound isassumed to bind to a CLK protein kinase polypeptide and block theability of CLK protein kinase to bind and/or turn over a substrate. If acompound enhances phosphorylation, the compound is assumed to bind to aCLK protein kinase polypeptide and facilitate the ability of CLK proteinkinase to bind and/or turn over a substrate.

[0073] The method can utilize any of the molecules disclosed in theinvention. These molecules include nucleic acid molecules encodingmCLK2, mCLK3, or mCLK4 polypeptides, nucleic acid vectors, recombinantcells, polypeptides, or antibodies of the invention.

[0074] Another aspect of the invention is a method of identifyingcompounds useful for the diagnosis or treatment of an abnormal conditionin an organism. The abnormal condition can be associated with anaberration in a signal transduction pathway characterized by aninteraction between a CLK protein kinase polypeptide and a naturalbinding partner. The method comprises the following steps: (a) adding acompound to cells; and (b) detecting whether the compound promotes ordisrupts said interaction between a CLK protein kinase polypeptide and anatural binding partner.

[0075] The term “abnormal condition” refers to a function in anorganism's cells or tissue that deviate from a normal function in thecells or tissue of that organism. In the context of this aspect of theinvention, abnormal conditions can be associated with cell proliferationor with RNA splicing.

[0076] Aberrant cell proliferative conditions include cancers such asfibrotic and mesangial disorders, abnormal angiogenesis andvasculogenesis, wound healing, psoriasis, diabetes mellitus, andinflammation.

[0077] RNA splicing is a necessary function of a cell that occurs in acell nucleus. This process is the last step in the synthesis of messageRNA from DNA. One molecule of RNA transcribed from DNA is tied into alariat, incised in at least two places at the intersection of thestrands, the lariat is excised, and the non-excised portion is ligatedtogether. The modified RNA is then fit to be message RNA and is ejectedfrom the cell nucleus to be translated into a polypeptide. Thus anyaberrations that exist in an organisms ability to splice the RNA of aparticular gene could result in the deficiency of a cellular agent andgive rise to an abnormal condition.

[0078] Thus, regulating RNA splicing could be useful in treating cancer.For example, it is known that proteins such as Raf or src becomeoncogenic when made in a truncated form, such as could happen when RNAis incorrectly spliced. For this reason, the proteins of the inventionmight be useful for finding compounds to treat cancer. In addition,molecules involved in RNA processing have been linked tospermatogenesis. Thus, modifying RNA processing could lead to more sperm(to treat infertility) or less sperm. These methods would preferablyinvolve CLK3 due to its high expression in the testis.

[0079] The abnormal condition can be diagnosed when the organism's cellsexist within the organism or outside of the organism. Cells existingoutside the organism can be maintained or grown in cell culture dishes.For cells harbored within the organism, many techniques exist in the artto administer compounds, including (but not limited to) oral,parenteral, dermal, and injection applications. For cells outside of thepatient, multiple techniques exist in the art to administer thecompounds, including (but not limited to) cell microinjectiontechniques, transformation techniques, and carrier techniques.

[0080] The term “signal transduction pathway” refers to the moleculesthat propagate an extracellular signal through the cell membrane tobecome an intracellular signal. This signal can then stimulate acellular response. The polypeptide molecules involved in signaltransduction processes are typically receptor and non-receptor proteinkinases, receptor and non-receptor protein phosphatases, andtranscription factors.

[0081] The term “aberration”, in conjunction with a signal transductionprocess, refers to a CLK protein kinase polypeptide that is over- orunder-expressed in an organism, mutated such that its catalytic activityis lower or higher than wild-type CLK protein kinase, mutated such thatit can no longer interact with a binding partner, is no longer modifiedby another protein kinase or protein phosphatase, or no longer interactswith a binding partner.

[0082] The term “interaction” defines the complex formed between a CLKprotein kinase polypeptide and a natural binding partner. Compounds canbind to either the CLK protein kinase polypeptide or the natural bindingpartner and disrupt the interaction between the two molecules. Themethod can also be performed by administering a group of cellscontaining an aberration in a signal transduction process to an organismand monitoring the effect of administering a compound on organismfunction. The art contains multiple methods of introducing a group ofcells to an organism as well as methods of administering a compound toan organism. The organism is preferably an animal such as a frog, mouse,rat, rabbit, monkey, or ape, and also a human.

[0083] Methods of determining a compound's effect of detecting aninteraction between CLK serine/threonine kinases and natural bindingpartners exist in the art. These methods include, but are not limitedto, determining the effect of the compound upon the catalytic activityof a CLK protein kinase polypeptide, the phosphorylation state of theCLK protein kinase polypeptides or natural binding partners, the abilityof a CLK protein kinase to bind a natural binding partner, or adifference in a cell morphology.

[0084] Differences in cell morphology include growth rates,differentiation rates, cell hypertrophy, survival, or prevention of celldeath. These phenomena are-simply measured by methods in the art. Thesemethods can involve observing the number of cells or the appearance ofcells under a microscope with respect to time (days).

[0085] Another aspect of the invention relates to a method of diagnosingan abnormal condition associated with cell proliferation or RNA splicingin an organism. The abnormal condition can be associated with anaberration in a signal transduction pathway characterized by aninteraction between a CLK protein kinase polypeptide and a naturalbinding partner. The method comprises the step of detecting the abnormalinteraction.

[0086] The abnormal interaction can be assessed by the methods describedabove in reference to the identification of compounds useful fordiagnosing an abnormal condition in an organism.

[0087] In a final aspect, the invention features a method ofadministering a compound to a male organism that acts a contraceptive toreproduction. The compound can inhibit the catalytic activity of a CLKprotein kinase or inhibit the binding of a natural binding partner to aCLK protein kinase;

[0088] Preferred embodiments of the methods of the invention relate toCLK serine/threonine kinases that are isolated from mammals, preferablyhumans, and to organisms that are mammals, preferably humans.

[0089] The summary of the invention described above is not limiting andother features and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

[0090]FIG. 1 compares amino acid sequences encoded by mCLK1, mCLK2,mCLK3, and mCLK4 nucleic acid molecules cloned from mouse cells. Eachamino acid sequence is encoded between a start codon and a stop codonfrom its respective nucleic acid molecule. Dots indicate identical aminoacids and hyphens are introduced for optimal alignment. The predictednuclear localization signals are underlined. Invariant amino acidssignifying CDC2 like kinases are printed in bold. The catalytic domainis indicated by arrows. The LAMMER signature is indicated by asterisks.

[0091]FIG. 2 depicts an amino acid sequence of mCLK2.

[0092]FIG. 3 depicts a nucleic acid sequence of mCLK2.

[0093]FIG. 4 depicts an amino acid sequence of mCLK3.

[0094]FIG. 5 depicts a nucleic acid sequence of mCLK3.

[0095]FIG. 6 depicts an amino acid sequence of mCLK4.

[0096]FIG. 7 depicts a nucleic acid sequence of mCLK4.

DETAILED DESCRIPTION OF THE INVENTION

[0097] The present invention is based in part upon the isolation andcharacterization of nucleic acid molecules encoding novel proteinkinases designated mCLK2, mCLK3, and mCLK4. The invention also relatesto nucleic acid molecules encoding portions of these protein kinasepolypeptides, nucleic acid molecules encoding at least one mCLK2, mCLK3,or mCLK4 functional region, nucleic acid vectors harboring such nucleicacid molecules, cells containing such nucleic acid vectors, purifiedpolypeptides encoded by such nucleic acid molecules, antibodies to suchpolypeptides, and methods of identifying compounds that bind CLKserine/threonine kinases or abrogate their interactions with naturalbinding partners. Also disclosed are methods for diagnosing abnormalconditions in an organism with CLK protein kinase related molecules orcompounds. The invention also concerns using a CLK protein kinaserelated molecule or compound as a contraceptive to reproduction in amale organism.

[0098] The present invention discloses the discovery of the proteinkinases, mCLK2, mCLK3, and mCLK4. The predicted molecular weights of theencoded proteins are 59.9 kDa (mCLK2), 58.5 kDa (mCLK3), and 57.2 kDa(mCLK4).

[0099] As illustrated in FIG. 1, mCLK1, mCLK2, mCLK3, and mCLK4 sharethe essential features identifying them as LAMMER kinases. Yun et al.,1994, Genes. Dev. 8:1160-1173. They contain a nuclear localizationsignal (Dingwall and Laskey, 1991, Trends Biochem. Sci. 16:478-481), aswell as an unusually basic amino terminus composed of many serine andarginine residues. These serine and arginine amino acids likely embody asignal sequence localizing the protein to nuclear speckles. Hedley etal., 1995, Proc. Natl. Acad. Sci. USA 92:11524-11528; Colwill et al.,1996, EMBO J. 15:265-275. The amino terminus is the most divergentportion of the proteins, suggesting that this area could containinformation specific to each protein. The catalytic domain is homologousamong all family members, with only few amino acid changes. Furthermore,all amino acids known to define the subfamily of CDC2 like kinases arepresent in all four proteins. Ben-David et al., 1991, EMBO J.10:317-325.

[0100] mCLK1 has been shown to interact with ASF/SF2, SRp2O and hnRNPproteins in a yeast two hybrid system. Because hnRNP-K binds to theprotooncogene p95^(vav), mCLK1 could be implicated in transmittingsignals that regulate the expression of the protooncogenes myc and fosin hematopoietic cells. Thus the role of CLK serine/threonine kinasesmay not be limited to simply maintaining RNA splicing and translocationevents in the cell; CLK serine/threonine kinases may also be linked toregulating the flow of extracellular signals within hematopoietic cells.In addition, CLK serine/threonine kinases may be targets for compoundsthat could ameliorate cancers associated with uncontrolled regulation ofthe protooncogenes p₉₅ ^(vav), myc, and fos. Because over-expression ofCLK serine/threonine kinases themselves have been implicated in certaintypes of cancer cell lines, compounds that inhibit their catalyticactivity or disrupt their interactions with natural binding partners mayact as anti-cancer therapeutics.

[0101] Even though CLK serine/threonine kinases other than mCLK2, mCLK3,and mCLK4 have been described previously, the methods of the inventionrelate to CLK serine/threonine kinases in general as the methodsdescribed herein are not disclosed elsewhere. Thus the methods of theinvention include antibodies and other compounds with specific bindingaffinity to mCLK2, mCLK3, and mCLK4 as well as antibodies and othercompounds that interact with other CLK protein kinase polypeptides.

[0102] Various other features and aspects of the invention are: nucleicacid molecules encoding a mCLK2, mCLK3, or mCLK4 polypeptide; nucleicacid probes for the detection of CLK serine/threonine kinases; aprobe-based method and kit for detecting CLK protein kinase messages inother organisms; DNA constructs comprising a mCLK2, mCLK3, or mCLK4nucleic acid molecule and cells containing these constructs; purifiedmCLK2, mCLK3, or mCLK4 polypeptides; mCLK2, mCLK3, or mCLK4 antibodiesand hybridomas; antibody-based methods and kits for detecting CLKserine/threonine kinases; identification of agents; isolation ofcompounds which interact with a CLK protein kinase polypeptide;compositions of compounds that interact with CLK serine/threoninekinases; pharmaceutical formulations and modes of administration;derivatives of complexes; antibodies to complexes; disruption of CLKprotein kinase protein complexes; purification and production ofcomplexes; transgenic animals containing mCLK2, mCLK3, or mCLK4 nucleicacid constructs; antisense and ribozyme approaches, gene therapy; andevaluation of disorders one skilled in the art would note that aderivative of a complex can manifest itself as a derivative of any oneof the molecules in that complex, including derivatives of a nucleicacid molecule, a polypeptide, or a compound bound to a polypeptide. Allof these aspects and features are explained in detail with respect tothe protein PYK-2 in PCT publication WO 96/18738, which is incorporatedherein by reference in its entirety, including any drawings. Thoseskilled in the art will readily appreciate that such descriptions can beeasily adapted to mCLK2, mCLK3, mCLK4, or other CLK serine/threoninekinases as well, and is equally applicable to the present invention.

[0103] Other features and aspects of the invention are depicted in PCTApplication WO 97/48723 (PCT/IB97/00946), published on Dec. 24, 1997,filed on Jun. 17, 1997, Axel Ullrich et al., entitled “Novel PTP20,PCP-2, BDP-1, CLK, and SIRP Proteins and Related Products and Methods,”hereby incoroporated by reference herein in its entirety, including allfigures, tables, and drawings.

EXAMPLES

[0104] The examples below are non-limiting and are merely representativeof various aspects and features of the present invention. The examplesbelow demonstrate the isolation, and characterization of the novelprotein kinases, mCLK2, mCLK3, and mCLK4.

Example 1 PCR Amplification and Cloning

[0105] The catalytic domain of protein kinases contains highly conservedregions, which have been successfully used to PCR amplify and clonenovel family members from a variety of species and tissues. Thesignature sequence HRDLAAR in the catalytic subdomain VI andD(V/M)WS(Y/F)G in subdomain IX were used to create degenerateoligonucleotides. Ciossek et al., 1995, Oncogene 11:2085-2095. Theseprimers were then used to search for unknown protein kinases involved inmuscle cell differentiation using reverse transcriptase PCR of totalRNA, isolated from various in vitro differentiated stages of the mousemyoblast cell line C2C12. Lechner et al., 1996, Proc. Natl. Acad. Sci.USA 93:4355-4359.

[0106] From the approximately 300 fragments which were sequenced one wasnovel. It derived from a member of the LAMMER family of dual specificitykinases (Yun et al., 1994, Genes. Dev. 8:1160-1173), also known as CLKkinases (Ben-David et al., 1991, EMBO J. 10:317-325) or STY (Howell etal., 1991, Mol. Cell. Biol. 11:568-572) and shared a high homology to apart of the human cDNA hCLK2. To obtain the full length clone and tosearch for other closely related sequences, a mouse 11.5 p.c. embryoniclibrary was screened at low stringency using the original 250 bp PCRfragment as a probe. Three highly related full-length cDNA sequencesdefining different members of the CLK family using this technique.

[0107] The same libraries were rescreened with a mixture of mCLK1, 2, 3,and 4 fragments at low stringency to isolate additional novel members ofthis family. Reverse transcriptase PCR reactions were performed onbrain, kidney and liver poly (A)⁺ RNA with degenerate primers coding forthe DLKPEN (SEQ ID NO. 1) and AMMERI (SEQ ID NO. 2) motifs. Theseefforts did not identify additional genes.

[0108] Reverse transcriptase PCR reactions were performed with 2 μg oftotal RNA prepared from confluent or differentiated (day 7) mouse C2C12myoblasts (Lechner et al., 1996, Proc. Natl. Acad. Sci. USA93:4355-4359) using degenerate oligonucleotide primers. Ciossek et al.,1995, Oncogene 11:2085-2095. Briefly, 2 μg of RNA were reversetranscribed in the presence of 1 μM degenerate antisense primer, 250 μMof each nucleotide and 75 units of Stratascript reverse transcriptase(Stratagene) in a total volume of 20 μl for 30 min at 42° C. 2 μl of theabove reaction was used in a PCR reaction using degenerate sense andantisense oligonucleotides (1 μM each), 25 μM of each nucleotide and 2.5units Taq polymerase (Boehringer). 30 cycles were performed with 1 minfor each 94° C., 50° C. and 72° C. step. Fragments of approximately 250bp were gel purified, cloned in Bluescript and sequenced.

[0109] mCLK2, mCLK3 and mCLK4 were cloned from a mouse embryo 11.5 p.c.1ZAP cDNA library (Ciossek et al., 1995, Oncogene 11:2085-2095) usingthe isolated PCR fragment as a probe according to manufacturer'sinstructions (final wash in 0.5×SSC/0.1% SDS at 42° C.) (Stratagene).mCLK1 was cloned by reverse transcriptase PCR from 1 μg brain poly (A)⁺RNA using specific primers mCLKls-Bam, CGGGATCCCTTCGCCTTGCAGCTTTGTC (SEQID NO. 3) and mCLKlas-EcoRI, CGGAATTCCTAGACTGATACAGTCTGTAAG (SEQ ID NO.4), and Pwo polymerase (Boehringer).

[0110] DNA sequencing was performed using the dideoxynucleotide chaintermination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA74:5463-5467) using sequenase, reagents and protocols supplied by UnitedStates Biochemical Corporation. Comparisons of the deduced proteinsequences were carried out using MacDNASIS PRO (Hitachi) software. Aminoacid alignments were constructed using a Waterman algorithm.

Example 2 Tissue Distribution of CLK Serine/Threonine Kinases

[0111] Expression patterns of CLK kinase genes (including the previouslycloned mCLK1) were analyzed by Northern blot hybridization of total RNAfrom selected mouse tissues, as well as from different mouse tumor celllines. Each CLK gene was detected in all investigated tissues, althoughthe expression patterns were different for each gene. The expected sizeof the full-length mRNA is ˜1.8 kb for all CLK kinases, and this wasdetected in all tissues and cell lines, albeit at different levels.

[0112] A doublet was detected at around 1.8 kb, whereby the upper bandrepresents the message of the full length protein and the lower one islikely to be the alternatively spliced form, responsible for atruncated, catalytically inactive protein. Duncan et al., 1995, J. Biol.Chem. 270:21524-21531. Commensurate with this alternative splice,differences in the ratio of the two alternatively spliced messages weredetected for each CLK gene.

[0113] Differences in expression patterns were observed for the CLKgenes, especially in testes. Low mCLK1 expression levels were observedin testes as compared to mCLK2, mCLK3 and mCLK4. However, while almostall of the mCLK3 message represented the catalytically active spliceform, mCLK4 was expressed predominantly as a message encoding thetruncated protein. mCLK2 was also highly expressed in this tissue, butas a larger transcript. Similar large transcripts, which did notcorrespond to the expected message size, were detected for all mCLKgenes which most likely represented non- or partially spliced messagesin analogy to mCLK1. Duncan et al., 1995, J. Biol. Chem.270:21524-21531. The ratio of these larger RNA species, when compared tothe coding mRNA, varied among the CLK kinases.

[0114] Because it was reported (Ben-David et al., 1991, EMBO J.10:317-325) that mCLK1 kinase was over-expressed in certain cancer celllines, studies were extended to mCLK1-4. Although messages for the fourgenes were detected in all cell lines tested, albeit in sometimes verylow quantities, significant differences of expression levels between thecell lines for each individual gene were observed. However, an overallincrease of mCLK mRNA was not detected in transformed cells, even thoughhigher levels of particular mCLK messages were detected in some cell.Low expression levels were detected in Hybridoma, WEHI and NF561 celllines, with the majority of the messages representing the splice formencoding the truncated product. The mRNA expression levels of mCLK1-4genes were investigated in the C2C12 cell line and Li adipocytes duringdifferentiation, but no noticeable change in expression was detected.

[0115] RNA was extracted from frozen adult mice tissues or tissueculture cells. Puissant and Houdebine, 1990, Biotechniques 8:148-149. 10μg total RNA was then electrophoresed in 1.2% agarose formaldehyde gels(Sambrook et al., 1989, Cold Spring Harbour Laboratory Press) andtransferred to Hybond N membranes (Amersham). Hybridization wasperformed overnight in 50% formamide, 5×SSC (750 mM sodium chloride, 75mM sodium citrate), 5×Denhardt's (0.1% Ficoll 400, 0.1%polyvinylpyrrolidone, 0.1% BSA), 0.2% SDS and 100 μg/ml salmon spermDNA. 1-3×10⁶ cpM/ml of ³²p-random primed DNA probe (Amersham Megaprimekit) was used, followed by washes at 0.2×SSC/0.1% SDS at 42° C. Blotswere incubated with Hyperfilm-MP (Amersham) at −80° C. for 2 weeks.Membranes were stripped for reuse by boiling in 0.1% SDS/water.

Example 3 Expression of Functionally Active CLK Protein Kinases

[0116] Glutathione S-transferase (GST) mCLK1-4 fusion constructs weregenerated to investigate the catalytic activity of these proteinkinases. These protein kinases were cloned from pcDNA and expressed invitro. The expression levels were almost identical and full-lengthfusion proteins of the expected molecular weights were obtained. The GSTfusion proteins were purified on glutathione-sepharose beads andutilized to perform in vitro kinase assays using myelin basic protein orhistone H1 as substrates. All constructs were catalytically active,autophosphorylated, and the levels of activity were clearly above thebackground seen from an equivalent amount of in vitro produced GSTprotein alone.

[0117] Catalytically inactive lysine to arginine mutants could notphosphorylate any substrate above background phosphorylation. However,mCLK1 and mCLK4 displayed a dramatic difference in enzymatic activityversus mCLK2 and mCLK3. This observation was consistent even whenchanging a variety of buffer conditions during fusion proteinpurification and assays or when changing metal ion concentrations.Several fold changes in kinase activity were observed due to theconditions used, but differences in enzymatic activities seen betweenthe two groups of mCLK kinases persisted.

[0118] Phosphorylation specificity of mCLK1-4 protein kinases wereexamined and compared using biochemically purified and dephosphorylatedSR proteins as substrates. SR proteins were purified from 5×10⁹ logphase F-MEL suspension cells according to standard procedures. Analiquot of the purified proteins was analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) to confirm that theexpected proteins were purified to near homogeneity.

[0119] Following dephosphorylation by the protein phosphatase 1 gcatalytic subunit, SR proteins were used as substrates for the in vitroproduced and purified GST-mCLK1-4 fusion proteins in an in vitro kinaseassay. All mCLK kinases were able to phosphorylate SRp2O, SRp3Oa and toa lesser extent SRp4O and SRp55. The lower signal of SRp4O and SRp55relative to SRp2O and SRp3O most likely reflected the lower quantity ofthese proteins. SRp75 was not visualized in these experiments since theautophosphorylated mCLK proteins migrated at the same position. mCLK1and mCLK4 phosphorylated SRp3Oa (upper band) more strongly than SRp3Ob,whereas mCLK2 and mCLK3 phosphorylated both with almost equalefficiency. A marked difference in catalytic activity was visualizedbetween mCLK1 and mCLK4 versus mCLK2 and mCLK3, despite equal amounts ofprotein in each assay.

[0120] To investigate the specificity of mCLK kinases, recombinant humanpeptidyl-prolyl isomerase PIN1 was utilized as a substrate. Lu et al.,1996, Nature 380:544-547. Although it also contains several aminoterminal serine and arginine residues and is localized to nuclearspeckles, neither of the mCLK kinases was able to phosphorylate thisprotein in vitro.

[0121] GST fusion constructs were generated by subcloning full lengthmCLK1, mCLK2, mCLK3 and mCLK4 cDNAs by PCR into pGEX vectors(Pharmacia), creating in-frame glutathione S-transferase (GST) fusionconstructs using the-following primers for PCR: mCLKls-Bam (as above);mCLK1as-Not I, TATAGCGGCCGCTAGACTGATACAGTCTGT; (SEQ ID NO. 5) mCLK2s-SmaI, TCCCCCGGGATGCCCCATCCCCGAAGGTACCA; (SEQ ID NO. 6) mCLK2as-Not I,TATAGCGGCCGCTCACCGACTGATATCCCGACTGGAGTC; (SEQ ID NO. 7) mCLK3s-Sma I,TCCCCCGGGGAGACGATGCATCACTGTAAG; (SEQ ID NO. 8) mCLK3as-Not I,TATAGCGGCCGCGCTGGCCTGCACCTGTCATCTGCTGGG; (SEQ ID NO. 9) mCLK4s-EcoRI,CGGAATTCATGCGGCATTCCAAACGAACTC, (SEQ ID NO. 10) mCLK4as-Not I,TATAGCGGCCGCCCTGACTCCCACTCATTTCCTTTTTAA. (SEQ ID NO. 11)

[0122] The cDNAs encoding the fusion construct were then recloned inpcDNA3 (Invitrogen) by PCR using the GST upstream primers: GST-EcoRI,CGGAATTCCGCCACCATGGCCCCTATACTAGGTTAT (SEQ ID NO. 12) (for mCLK1) andGST-Hind III, GCCAAGCTTGCCACCATGGCCCCTATACTAGGTTAT (SEQ ID NO. 13) (formCLK2, mCLK3 and mCLK4).

[0123] Integrity of the clones was checked by sequencing and by acoupled transcription-translation assay using T7 RNA polymerase andrabbit reticulocyte lysate according to the manufacturer's protocol(Promega).

[0124] mCLK 1-4 mutants containing a lysine (K) to arginine (R)substitution at position 190 (mCLK1), 192 (mCLK2), 186 (mCLK3) and 189(mCLK4) were generated using a site-directed mutagenesis protocol.Kunkel, 1985, Proc. Nati. Acad. Sci. USA 82:488-492. Oligonucleotideprimers were as follows: (mCLK1-K190R) GTAGCAGTAAGAATAGTTAAA;(mCLK2-K192R) (SEQ ID NO. 14) GTTGCCCTGAGGATCATTAAGAAT; (mCLK3-K186R)(SEQ ID NO. 15) GTTGCCCTGAGGATCATCCGGAAT; (mCLK4-K189R) (SEQ ID NO. 16)TACAATTCTCACTGCTACATGTAAGCCATC (SEQ ID NO. 17).

[0125]³⁵S-methionine labeled GST-mCLK1-4 fusion proteins were producedin a 50 gl coupled in vitro transcription/translation reaction usingmanufacturer's instructions (Promega).

[0126] 2 gl of each lysate was checked and quantified for the amounts ofproduced protein by SDS-PAGE and autoradiography. Equal amounts (usually20-30, μl of lysate) were added to 500 μl PBS (1 mM PMSF, 10 μg/mlaprotinine), 30 μl of GSH-sepharose beads (Pharmacia) and incubated on arotating wheel for 2 hours at 4° C. This step resulted in quantitativebinding of the fusion proteins. The beads were then washed three timesin 500 μl PBS and once in 500 μl kinase assay buffer (20 mM Hepes, 10 mMMgCl₂, 1 mM DTT, 200 μM sodium orthovanadate, 1 mM EGTA, pH 7.5). Theassay was carried out for 30 minutes at room temperature in 30 μl kinaseassay buffer with 20 μM ATP, 4 μCi γ-³²P-ATP (Amersham, 10 mCi/ml) and14.5 μg of myelin basic protein (Sigma) or histone H1 (Boehringer)respectively.

[0127] SR protein kinase assays were essentially carried out asdescribed above, except that ˜2.5 μg of dephosphorylated SR proteinswere used and that the kinase assay buffer also contained 1 μMMicrocystin LR (Sigma). The reaction was stopped by adding 30 μl of2×SDS sample buffer. The samples were boiled for 5 minutes and 15 μlwere loaded on a 15% SDS-polyacrylamide gel. Following electrophoresis,the gels were stained with Coomassie, dried and exposed to Hyperfilm-MP(Amersham) for 24 hours. The ³⁵S-methionine signal was suppressed with a3M Whatman paper placed between the film and the gel.

[0128] SR proteins were purified from 5×10⁹ Friend murineerythroleukemia cells (F-MEL) according to the protocol described(Zahler et al., 1992, Genes Dev 6:837-847) and resuspended in buffer D.Dignam et al., 1983, Nucleic Acids Res. 11:1475-1489. 30 μl of SRproteins (˜0.5 μg/μl) were incubated on ice for 10 minutes in 0.7 mMMnCl₂ and 5 mU Protein Phosphatase 1 g-catalytic subunit (Boehringer),followed by 60 minutes at 30□C. Mermoud et al., 1994, EMBO J.13:5679-5688. 5 μl of dephosphorylated SR proteins were used per assay.

Example 4 Nucleic Acid Probes, Methods, and Kits for Detection of CLKKinases and Other Related Polyoeptides

[0129] A nucleic acid probe of the present invention may be used toprobe an appropriate chromosomal or cDNA library by usual hybridizationmethods to obtain other nucleic acid molecules of the present invention.A chromosomal DNA or cDNA library may be prepared from appropriate cellsaccording to recognized methods in the art (cf. “Molecular Cloning: ALaboratory Manual”, second edition, Cold Spring Harbor Laboratory,Sambrook, Fritsch, & Maniatis, eds., 1989).

[0130] In the alternative, chemical synthesis can be carried out inorder to obtain nucleic acid probes having nucleotide sequences whichcorrespond to N-terminal and C-terminal portions of the amino acidsequence of the polypeptide of interest. The synthesized nucleic acidprobes may be used as primers in a polymerase chain reaction (PCR)carried out in accordance with recognized PCR techniques, essentiallyaccording to PCR Protocols, “A Guide to Methods and Applications”,Academic Press, Michael, et al., eds., 1990, utilizing the appropriatechromosomal or cDNA library to obtain the fragment of the presentinvention.

[0131] One skilled in the art can readily design such probes based onthe sequence disclosed herein using methods of computer alignment andsequence analysis known in the art (“Molecular Cloning: A LaboratoryManual”, 1989, supra). The hybridization probes of the present inventioncan be labeled by standard labeling techniques such as with aradiolabel, enzyme label, fluorescent label, biotin-avidin label,chemiluminescence, and the like. After hybridization, the probes may bevisualized using known methods.

[0132] The nucleic acid probes of the present invention include RNA, aswell as DNA probes, such probes being generated using techniques knownin the art. The nucleic acid probe may be immobilized on a solidsupport. Examples of such solid supports include, but are not limitedto, plastics such as polycarbonate, complex carbohydrates such asagarose and sepharose, and acrylic resins, such as polyacrylamide andlatex beads. Techniques for coupling nucleic acid probes to such solidsupports are well known in the art.

[0133] The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The samples used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample that is compatiblewith the method utilized.

[0134] One method of detecting the presence of nucleic acids of theinvention in a sample comprises (a) contacting said sample with theabove-described nucleic acid probe under conditions such thathybridization occurs, and (b) detecting the presence of said probe boundto said nucleic acid molecule. One skilled in the art would select thenucleic acid probe according to techniques known in the art as describedabove. Samples to be tested include but should not be limited to RNAsamples of human tissue.

[0135] A kit for detecting the presence of nucleic acids of theinvention in a sample comprises at least one container means havingdisposed therein the above-described nucleic acid probe. The kit mayfurther comprise other containers comprising one or more of thefollowing: wash reagents and reagents capable of detecting the presenceof bound nucleic acid probe. Examples of detection reagents include, butare not limited to radiolabelled probes, enzymatic labeled probes(horseradish peroxidase, alkaline phosphatase), and affinity labeledprobes (biotin, avidin, or streptavidin). Preferably, the kit furthercomprises instructions for use.

[0136] A compartmentalized kit includes any kit in which reagents arecontained in separate containers. Such containers include small glasscontainers, plastic containers or strips of plastic or paper. Suchcontainers allow the efficient transfer of reagents from one compartmentto another compartment such that the samples and reagents are notcross-contaminated and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers include a container that accepts the test sample, a containerthat contains the probe or primers used in the assay, containers thatcontain wash reagents (such as phosphate buffered saline, Tris-buffers,and the like), and containers that contain the reagents used to detectthe hybridized probe, bound antibody, amplified product, or the like.One skilled in the art will readily recognize that the nucleic acidprobes described in the present invention can readily be incorporatedinto one of the established kit formats that are well known in the art.

[0137] One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art which are encompassed within the spirit ofthe invention are defined by the scope of the claims.

[0138] It will be readily apparent to one skilled in the art thatvarying substitutions and modifications may be made to the inventiondisclosed herein without departing from the scope and spirit of theinvention.

[0139] All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains.

[0140] The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of” and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description

1-6. (Cancelled).
 7. An isolated, enriched or purified mCLK2,polypeptide.
 8. The isolated, enriched, or purified mCLK2, polypeptide,wherein said polypeptide is a unique fragment comprising at leastseventeen contiguous amino acids present in the full-length amino acidsequence set forth in FIG. 1 and FIG.
 2. 9-18. (Cancelled).